snp haplotypes as diagnostic markers shrish tiwari ccmb, hyderabad
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SNP Haplotypes as Diagnostic Markers
Shrish TiwariCCMB, Hyderabad
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Introduction Genomic variation mostly (90%)
as single nucleotide differences (2/3 of which are transitions)
Non-uniformly distributed (on an average 1 difference for every 1000 bp)
Useful as genetic markers Unambiguous assay techniques Slow mutation rate Amenable for high-throughput
genotyping
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...C C ...C C AA T T G A C... T T G A C...
...C C ...C C GG T T G A C... T T G A C...
……G G G G TT A A C T G... A A C T G...
……G G G G CC A A C T G... A A C T G...
Single base-pair differences occurring in a population with a frequency of >1%
Single Nucleotide Polymorphism
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Types of SNPs
SNPs can occur in protein coding or non-coding DNA
The coding region SNPs can either be in exons (cSNPs), introns or regulatory regions
cSNPs may either be silent or cause a change in amino acid
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SNP databases dbSNP: (~3 million SNPs)
http://www.ncbi.nih.gov/SNP/
JSNP: (~195,000 SNPs) http://snp.ims.u-tokyo.ac.jp/
The SNP Consortium (TSC): http://snp.cshl.org/
Human Genome Variation Database http://hgvbase.cgb.ki.se/
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SNP analysis
SNP discovery: by sequencing, by EST sequence comparison
SNP validation: SNAPShot, DHPLC, Sequenom
SNP allele frequency
SNP association
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SNP discovery: Experimental approach Obtain DNA sequence around SNP
PCR amplify the segment of interest
Identify SNP by sequencing the segment from different individuals
Map the marker onto chromosome
Compute the allele frequency in population
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SNP discovery: in silico Align EST’s to themselves or to
cDNAs
Find variations in ESTs that align with 95% identity over >100bp
Distinguish true SNPs from sequencing errors by checking the base quality using Phred/Phrap set of programs
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The PolyBayes Program Use genomic sequence as reference
cluster and align all available EST sequences
remove repeats/paralogs
Use Bayesian statistics to distinguish polymorphic sites from
artifacts estimate likelihood
Marth, GT, Korf, I, Yandell, MD, Yeh, RT, Gu, Z, Zakeri, H, Stitziel, NO, Hillier, L, Kwok, P-Y, Gish, WR: A general approach to single-nucleotide polymorphism discovery. Nature Genet. 1999; 23:452-456.
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SOME OTHER SNP PREDICTION & SNP FINDING SOFTWARE
SEAN: Search for localized SNPs
and predict SNPs
(http://zebrafish.doc.ic.ac.uk/Sean/)
SNPpipeline: Mine SNPs stored in gene
databases
SNP Finder: For analyzing user-submitted
trace data
(http://gai.nci.nih.gov/)
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SNP Applications
Disease diagnosis
Identification of factors for increased susceptibility to disease
May be responsible for the different responses of individuals to drugs
Will help in developing personalised medicines
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SNP Haplotypes
Sets of SNPs in linkage disequilibrium
SNP genotype data (4 loci): 00 01 01 11
Possible SNP haplotypes: 0001/0111 0011/0101
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SNP-Haplotype Association
DNA Sequence
GATATTCGTACGGA-TGATGTTCGTACTGAATGATATTCGTACGGA-TGATATTCGTACGGAATGATGTTCGTACTGAATGATGTTCGTACTGAAT
SN
P
SN
P
1 2
3
4
5 6
AG- 2/6(BLACK EYE)GTA 3/6(BROWN EYE)
AGA 1/6 (BLUE EYE)
Haplotypes
PhenotypeBLACK EYE
BROWN EYE
BLACK EYE
BLUE EYE
BROWN EYE
BROWN EYE
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Haplotype database
HapMap: http://www.hapmap.org/
The international HapMap Consortium, The International HapMap Project, Nature 426, 789-796 (2003)
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Why SNP Haplotypes?
More reliable than single SNPs as markers
Usually less in number than the number of SNPs in the set
Haplotype tagging SNPs further reduce genotyping costs
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Association study Association of allele and phenotype
Association can occur if Allele is in linkage disequilibrium with
the mutation responsible for the phenotype
Allele is responsible for the phenotype
For association study a control and a case population is necessary
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SNP haplotypes as diagnostic markers Identify SNPs for gene
associated with disease
Find the different sets of SNPs in a control population
Find different sets of SNPs in affected population
Look for unique sets of SNPs in affected population
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References
A.J. Brookes, The essence of SNPs, Gene 234, 177-186 (1999).
Pui-Yan Kwok and Zhijie Gu, Single Nucleotide Polymorphism libraries: why and how are we building them, Mol. Med. Today 5, 538-543 (1999).
SNPs Science Primer: http://www.ncbi.nlm.nih.gov/About/primer/snps.html